Recombineering in Corynebacterium glutamicum combined with optical nanosensors: a general strategy for fast producer strain generation

Abstract

Recombineering in bacteria is a powerful technique for genome reconstruction, but until now, it was not generally applicable for development of small-molecule producers because of the inconspicuous phenotype of most compounds of biotechnological relevance. Here, we establish recombineering for Corynebacterium glutamicum using RecT of prophage Rac and combine this with our recently developed nanosensor technology, which enables the detection and isolation of productive mutants at the single-cell level via fluorescence-activated cell sorting (FACS). We call this new technology RecFACS, which we use for genomic site-directed saturation mutagenesis without relying on pre-constructed libraries to directly isolate l-lysine-producing cells. A mixture of 19 different oligonucleotides was used targeting codon 81 in murE of the wild-type, at a locus where one single mutation is known to cause l-lysine production. Using RecFACS, productive mutants were screened and isolated. Sequencing revealed 12 different amino acid exchanges in the targeted murE codon, which caused different l-lysine production titers. Apart from introducing a rapid genome construction technology for C. glutamicum, the present work demonstrates that RecFACS is suitable to simply create producers as well as genetic diversity in one single step, thus establishing a new general concept in synthetic biology.

A standardized workflow for surveying recombinases expands bacterial genome‐editing capabilities

“…Our work challenges assumptions of species specificity based on these reports, instead suggesting promiscuity of oligonucleotide‐mediated recombinase activity. Such non‐specific activity has been documented in other bacteria, as in the case of Corynebacterium glutamicum (Binder et al ., 2013) and E. coli (Datta et al ., 2008). While the first study focused only on RecT of Rac prophage, in the second, authors detected high rates of recombineering activity in E. coli from an array of recombinases originating from both gram‐negative and gram‐positive bacteria (10 −4 to 10 −1 mutants/viable cell).…”

“…Recβ has only been shown to catalyze bacterial genomic recombination of foreign ssDNA in enterobacteriaceae (Nyerges et al ., 2016). In contrast, it fails to function in species such as P. syringae (Swingle et al ., 2010) and C. glutamicum (Binder et al ., 2013). Recβ activity in P. putida KT2440 has been documented at ~10 −7 mutants/viable cell (Luo et al ., 2016), a result equal to, if not lower than, basal recombineering rates in this organism.…”

“…Since its advent, MAGE recombineering has brought about a multitude of advances, including genome‐wide codon replacement, biosynthesis of lycopene and aromatic amino acids, and comprehensive mutant library generation (Wang et al ., 2009; Isaacs et al ., 2011; Gallagher et al ., 2014; Nyerges et al ., 2016). Unfortunately, β‐recombinase (Recβ) recombineering activity is not well‐conserved in bacterial species outside of the Enterobacteriaceae family (Van Kessel and Hatfull, 2008; Binder et al ., 2013; Lennen et al ., 2016); this limited conservation is thought to stem from specificity of host–phage recombinase interactions (Sun et al ., 2015). The apparent species specificity of recombineering systems has spurred a search for functional Recβ analogues in alternative lineages, with limited success in microbes including Salmonella , P. syringae, Corynebacteria , Lactobacilli and Mycobacteria (Van Kessel and Hatfull, 2008; Gerlach et al ., 2009; Swingle et al ., 2010; Pijkeren and Britton, 2012; Binder et al ., 2013; Oh and Pijkeren, 2014).…”

Involvement of the Global Regulator GlxR in 3-Hydroxybenzoate and Gentisate Utilization by Corynebacterium glutamicum

“…Derivative strains of Corynebacterium were used to produce 2.5 million tons of glutamate as monosodium glutamate per year as well as several thousand tons of other amino acids such as lysine, isoleucine, tryptophan, and threonine (16,21). Recently, C. glutamicum ATCC 13032 has been found to utilize a number of aromatic compounds as its sole carbon and energy source, including 3-HBA and GEN (22).…”

“…These results indicated that the overexpression of glxR repressed all three operons to significant degrees. Recently, a unique recombineering method for constructing a mutant strain has been established in Corynebacterium glutamicum to study its biological function in vivo (21). If the mutation of GlxR binding sites in the glxR overexpression strain had been performed using this method, the relationship between the effects of the binding site mutations on gene expression and the binding of GlxR would have been made much more direct.…”